The subject matter of the present invention is a method for determining an analyte present in a solution. More precisely, it relates to a method with which it is possible to detect and/or quantify this analyte. The analyte may be a chemical or biological entity.
Therefore, the present invention finds numerous applications in the area of chemical or biological analysis, for example for the determination of antigens, antibodies, DNA or RNA sequences, bacteria, viruses, bacterial fragments, etc.
Numerous systems for chemical or biological analyses are based on the reaction of the analyte to be determined with a suitable compound to form a reaction product, which must subsequently be detected and/or quantified in order to determine the presence and concentration of this analyte. Generally, this detection is made via labellers such as radioactive or luminescent tracers, enzymes or others which may be carried by the added compound added to react with the analyte to be detected. The use of such tracers assumes the use of specific apparatus to ensure the detection and/or quantification of the analyte.
Other detection and quantification methods such as topographic methods for example using atomic force microscopy (AFM), magnetic methods, electric methods for example by measuring capacity variation, and optic methods may also be used as described in FR-A-2 758 884 (1).
These methods, however, also have the disadvantage of requiring specific apparatus which can at times be costly.
The present invention sets out precisely to provide a method of determining an analyte which avoids the use of such apparatus and ensures the detection and/or quantification of the analyte using very conventional components at affordable price.
According to the invention, the method for determining an analyte in a solution comprises the following steps:
a) fixing and immobilizing said analyte on the inner surface of a conduit having a reduced cross section over all or part of it, and
b) determining the variation in load loss of a fluid circulating inside the conduit, due to the analyte which has been fixed and immobilized at least in the reduced cross section part of said conduit during step a).
The method of the invention is therefore based on the use of a conduit having a suitable cross section over all or part of it, in which, during step a), the analyte to be determined is fixed and immobilized.
The fixing and immobilization of the analyte is made at least over all or part of the reduced cross section of the conduit, but they may also be conducted over the entire conduit.
This fixing and immobilization may be made either by contacting the solution with the inner surface of the conduit (for example by dipping) or by circulating the solution in the conduit, this circulation possibly entailing at least one sequential stop.
During the subsequent step b), the variation in load loss of a fluid circulating in the conduit is determined, such variation resulting from fixation of the analyte and being related to the quantity of analyte fixed.
This quantification of the analyte may be conducted using pre-set plots or curves, or by experimentation, or by simulation on the basis of known geometric data on the conduit and on the analytes used (size and quantity). Therefore, the value of the load loss is related to the quantity of analyte immobilized in the conduit and/or the size of this analyte.
Through experimentation, it is possible to cross-refer the measurement obtained in the method of the invention to previously made measurements to calibrate the performance of the conduit.
With both these methods it is possible to plot graphs showing the relationship between load loss and the quantity of immobilized analyte. Therefore, via simple measurement of load loss, it is possible to quantify the level of analyte immobilized in the conduit during step a) of the method of the invention.
According to the invention, it is possible to determine the variation in load loss by measuring the difference in pressure, at constant flow rate between two points located either side of the reduced cross section of the conduit, of a fluid circulating in the conduit before carrying out step a) and after conducting step a) of the method, or else the difference in flow rate at constant pressure before and after step a).
The fluid used for this determination may be all or part of the initial solution (in this case, step a) and step b) are simultaneous or successive) or a different liquid to this solution.
In the prior art, measurement of load loss in a conduit with a circulating fluid was used to determine certain characteristics of the fluid, its viscosity for example, and consequently its content of a particular product able to modify its viscosity. In document U.S. Pat. No. 4,964,847 [2] for example, this measurement is used to estimate packed cell volumes in blood.
This type of determination method is different from the method of the invention in which detection and quantification of the analyte require its immobilization on the wall of a conduit having a suitable shape and having, in addition, the property of being able to fix the analyte to be determined.
This latter property may be conferred upon the conduit by fixing on its wall a ligand that is able to bind with the analyte.
In this case, the determination of the analyte involves a recognition reaction between the analyte and the ligand with formation of an analyte-ligand complex immobilized in the conduit.
In this embodiment of the method of the invention, prior to step a), the wall of the reduced cross section is coated over all or part with at least one ligand able to fix itself to the analyte.
In this case, the presence of the ligand can be detected by determining the variation in load loss through measurement of the difference in pressure at constant flow rate, or the difference in flow rate at constant pressure, between two points located either side of the reduced cross section of the conduit, of a fluid circulating inside the conduit before and after making the ligand coating.
According to one variant of embodiment of the invention, when the size of analyte to be determined, or of the complex formed through reaction of this analyte with a ligand, is too small to generate sufficient load loss in the reduced cross section part of the conduit, a physical or chemical reaction of the analyte is additionally performed with a support material to form an analyte-support conjugate of greater size than the analyte, this reaction taking place either when said analyte is free in the solution, before step a), or when the analyte is already fixed directly or indirectly onto the inner wall of the reduced cross section part of the conduit after step a) to form an analyte-support conjugate.
This reaction between the analyte and the support material may be conducted before adding the solution to the conduit, or before conducting step a), or after fixing the analyte on the wall of the reduced cross section part of the conduit, that is to say after step a) but before step b).
To promote the binding of the support material with the analyte, the latter generally comprises a ligand on its surface able to bind with the analyte.
According to the invention, by xe2x80x9canalytexe2x80x9d is meant any chemical or biological entity, in particular any biological entity in free form. As an illustration of analytes, the examples which may be cited are cells, organelles, viruses and bacteria, antibodies, antibody fragments, antigens, haptenes, lectines, sugars, ribonucleic and deoxyribonucelic acids, proteins, in particular A or G, hormones, hormone receptors, biotin, avidin, streptavidin and in general any molecule or macromolecule that is natural or synthetic, or analogue or even resulting from an association of at least two molecules or macromolecules of the type of those previously defined. Examples of associations of molecules or macromolecules which may be cited are the analyte-support conjugates which will be defined below.
According to the invention, by xe2x80x9csupport-materialxe2x80x9d is meant any type of biological, polymer, organic, inorganic or metallic support which may be distributed or dispersed in discrete form within a liquid medium, and most often, but not restrictively, in particle form.
Examples of support-materials of polymer type which may be cited are particles obtained by emulsion polymerization such as latex particles, or particles of greater size and grafted polymers, copolymers and polymers obtained by other means known to those skilled in the art.
Examples of support-materials of metallic type which may be cited are colloidal gold, ferromagnetic, ferrimagnetic, paramagentic or superparamagnetic particles, whether coated or not with natural or synthetic polymers which contain iron or other metals such as cobalt, nickel or others, either alone or in alloy form, whether magnetic or not.
Examples of support-materials of inorganic type which may be cited are particles containing silica or silicon, mica, glass and/or quartz.
Examples of support-materials of biological type which may be cited are mass proteins or those of greater size than the analyte to be determined in the free state.
By xe2x80x9canalyte-support conjugatexe2x80x9d is meant any analyte such as previously described, immobilized on a support-material such as previously defined by any means. Immobilization of the analytes on the support-material may be made by simple adsorption or via a chemical or physical reaction able to modify the surface of the support-material and therefore enabling fixing of the analyte by covalent bonds, or by chelation or by molecular recognition via a ligand immobilized on the support-material, and by any other conventional means able to withhold an analyte well known to those skilled in the art.
By xe2x80x9cligandxe2x80x9d is meant an element able, through a chemical or physical bond, to form a complex with an analyte.
Examples of ligands which may be cited are antibodies, antibody fragments, antigens, haptenes, lectines, sugars, ribonucleic acids, deoxyribonucleic acids, proteins in particular A or G, hormones, hormone receptors, biotin, avidin or streptavidin and, in general, natural or synthetic ligands and analogues of modified ligands, which may enter into competition with the ligands.
In the remaindes of this disclosure, xe2x80x9ccirculating cellxe2x80x9d will denote any type of system comprising the conduit with its reduced cross section part, and at least two inlet and outlet pipes allowing the passage of a fluid within the conduit.
By xe2x80x9creduced cross-sectionxe2x80x9d shall be meant a section which is generally narrower than that of the remainder of the conduit. In some cases, the entire conduit may have this reduced cross section. This portion of reduced cross section may also be called a xe2x80x9crestrictionxe2x80x9d and, owing to its particular analyte-fixing properties, it will be called hereinafter an xe2x80x9cactive zonexe2x80x9d.
This active zone is therefore formed of the wall of the conduit on which ligands may be fixed which are able to bind with the analyte.
These ligands are fixed on the wall by any means such as by chemical reaction (covalent bonding) or physical reaction (adsorption) to modify the surface of the wall and enable fixation of the ligand.
According to the invention, the reduced cross section part of the conduit able to fix and immobilize the analyte may be of different shapes. It may in particular be round, the conduit being of cylindrical shape, or rectangular when the fluid circulation will be plane-type circulation between two parallel surfaces.
The conduit may be made in various materials, for example in materials of polymer, inorganic or metal type.
Examples of polymer materials which may be cited are polystyrene-based polymers, polyacrylates, polymethacrylates, polybutadienes, polypropylenes, or others, either alone or in the form of copolymers.
Examples of inorganic materials which may be cited include silicon oxide, silicon nitride, silicon, glass and quartz.
According to the invention, the reduced cross section part of the conduit must be dimensioned such that it is compatible with the size of the analyte to be determined.
If the conduit is of large size in relation to the size of the immobilized analyte, the presence of this analyte will not lead to a significant variation in load loss. It will therefore not be possible either to detect or to quantify the analyte.
The theories of fluid mechanics show that in general the load loss xcex94P between the inlet and outlet of a portion of conduit having a reduced cross section is defined by the following equation:
xcex94P=KQ
where Q represents the volume flow of the liquid and K is a load loss coefficient related to the geometric characteristics of the conduit and the viscosity of the fluid.
According to their respective dimensions, the presence of analytes or analyte-support conjugates on the walls of the conduit causes a change in the load loss coefficient. With a conduit height close to the dimensions of the analyte or of the analyte-support conjugate, a variation in load loss coefficient is obtained which can be measured.
Therefore, to conduct the determination of the analyte under good conditions, a conduit is used whose reduced cross section part is of a size that is adapted to the size of the immobilized analyte or of the immobilized analyte-support conjugate.
Preferably, the smallest dimension Tc of the reduced cross section of the conduit is such that:
(Tc:3) less than TA or e less than Tc.
in which TA represents the size of the fixed analyte either as such or in the form of an analyte-support conjugate, on the wall of the reduced cross section part of the conduit, or e represents the thickness of the ligand.
When circulation of the fluid takes place between two parallel surfaces, Tc represents the distance between the two surfaces. In respect of a cylindrical conduit, Tc represents the diameter of the conduit.
The size of the analytes or analyte-support conjugates is very small since the analyte may be made up of one molecule or of one living cell; conduits of small dimensions are required, for example a few hundred nanometres or more.
Conduits having such dimensions may be obtained using microfabrication processes such as those used in microelectronics, micromechanics and, for example, using a method of the type described in FR-A-2 727 648 [3].
The method of the invention may be implemented in various manners, additionally conducting, if necessary, at least one conduit washing step between steps a) and b).
According to a first embodiment which may be used to determine analytes of sufficient volume, bacteria, viruses or bacteria fragments for example, the method entails the following steps:
1) contacting the solution containing the analyte with the active zone of the conduit to fix the analyte on this active zone,
2) washing the conduit to remove the part that has not reacted on this active zone, and
3) measuring the load loss in the conduit.
According to a second embodiment of the invention, intended to determine analytes of small size, the method entails the following steps:
1) reaction of the support-material with the analyte in the initial solution, to form the analyte-support conjugate in this solution,
2) contacting the solution with the active zone of the conduit to fix the analyte-support conjugate on this active zone,
3) washing the conduit to remove the part that has not reacted, and
4) measuring the load loss in the conduit.
According to a third embodiment of the invention, also intended to determine analytes of small size, the method entails the following steps:
1) contacting the solution containing the analyte with the active zone of the conduit to fix the analyte on this active zone,
2) washing the conduit to remove the part that has not reacted,
3) adding to the active zone of the conduit a solution containing a support-material to form the analyte-support conjugate directly on the active zone of the conduit,
4) washing the conduit to remove the support-material which has not reacted, and
5) measuring the load loss in the conduit.
Although it is preferable to use a liquid which does not contain any large molecule or biological cell to measure the load loss, it is nonetheless possible to consider using the liquids previously cited.
Therefore, according to one variant of these three embodiments, the washing step which precedes load loss measurement is omitted and this measurement is made using as liquid the solution added during the previous step, that is to say the analyte solution in the first embodiment, the analyte-support conjugate solution in the second embodiment, and the material-support solution in the third embodiment.
According to the invention, it is also possible to use the same basic principle, i.e. measurement of load loss induced by a product immobilized in a conduit, to determine the presence of a chemical or biological coating product on the inner surface of a conduit.
Therefore, a further purpose of the invention is a method to determine the presence of a chemical or biological coating on the inner surface of a conduit, which comprises the following steps:
1) placing the circulation fluid in the conduit,
2) measuring the load loss of said fluid in the conduit, and
3) determining, on the basis of measured load loss, the presence or non-presence of a coating in the conduit.
This method may be particularly useful to verify whether a ligand is in fact fixed and immobilized in the active zone of a conduit intended to determine an analyte using the method of the invention.
In this latter case, the dimensions of the conduit evidently need to be suitable to determine the load loss induced by the coating.
Preferably, the smallest dimension Tc of the conduit section is such that:
(Tc:3) less than e Tc
where e represents the thickness of the coating.
Other characteristics and advantages of the invention will become clearer on reading the following description, which is evidently given for illustrative purposes and is non-restrictive, with reference to the appended drawings.